Development and Validation of Discriminatory In-vitro Release Method for Intramammary Drug Product.

PURPOSE: Intramammary (IMM) formulations are locally acting and delivered intracisternally into the udder. No pharmacopeial in-vitro release method is available to differentiate between the IMM formulations. Our research aim is to develop in-vitro release methods that discriminate different IMM formulations (SPECTRAMAST® LC and in-house formulations). METHODOLOGY: Different in-house formulations were developed to simulate SPECTRAMAST® LC generics. SPECTRAMAST® LC and the in-house formulations were characterized for physicochemical attributes, such as particle size, rheology, drug content, sedimentation rate, and flocculation rate. The in-vitro release method was optimized by evaluating drug release using USP apparatuses 1, 2 (with and without enhancer/customized cells), and 4. Various test parameters, including medium effect (whole homogenized bovine milk versus aqueous buffer), medium volume (200-900 mL), and rotational speed (50-200 rpm) were investigated. RESULTS: Two potential in-vitro systems can be used as discriminatory methods for IMM formulations: USP apparatus 2 with the IMM formulation loaded into two containers a) customized formulation container (83.1 cm in height and 56.4 cm in width) or b) enhancer cells with their top adapted with mesh #40 (rotation speed:125 rpm and 900 mL of whole homogenized bovine milk). The release profile of SPECTRAMAST® LC at 1 h (99.8%) was not significantly different from formulations with similar physicochemical characteristics F-01 (99.1%) and F-02 (100.5%). Formulation with different physicochemical characteristics F-03 (44.3%) and F-04 (57.2%) showed slower release (1 h) than SPECTRAMAST® LC (98.8%). CONCLUSION: The developed in-vitro release methods can be used as a potential tool for in-vitro comparability evaluations for IMM formulations.

Nitroso Impurities in Drug Products: An Overview of Risk Assessment, Regulatory Milieu, and Control Strategy.

Many nitrosamines have been recognized to be carcinogenic for many decades. Despite the fact that several nitrosamine precursors are frequently used in the manufacturing of pharmaceutical products, their potential presence in pharmaceutical products has previously been overlooked due to a lack of understanding on how they form during the manufacturing process. From the risk assessment, it is clear that nitrosamines or their precursors may be present in any component of the finished dosage form. As a risk mitigation strategy, components with a high potential to form nitrosamine should be avoided. In the absence of suitable alternatives, sufficient measures to maintain nitrosamines below acceptable intake levels must be applied. Excipient manufacturing pathways must be extensively studied in order to identify probable excipient components that may contribute to nitrosamine formation. The manufacturers must not solely rely on pharmacopeial specifications for APIs and excipients, rather, they should also develop and implement additional strategies to control nitrosamine impurities. The formulation can be supplemented with nitrosating inhibitors, such as vitamin C, to stop the generation of nitrosamine. The purpose of this review is to identify key risk factors with regard to nitrosamine formation in pharmaceutical dosage forms and provide an effective control strategy to contain them below acceptable daily intake limits.

Pyrimethamine 3D printlets for pediatric toxoplasmosis: design, pharmacokinetics, and anti-toxoplasma activity.

OBJECTIVES: The focus of the present research is to develop printlet formulations of pyrimethamine (PMT). METHODS: Printlets formulation of PMT were developed by screening design by varying laser scanning speed, Kollidon® VA 64, polyvinylpyrrolidone, and disintegrant. RESULTS: Laser scanning speed, Kollidon® VA, and disintegrant had statistically significant effect on hardness, disintegration time, and/or dissolution (p < 0.05). Dissolution was almost 100% in 30 min. X-ray powder diffraction indicated partial amorphous transformation of the crystalline drug. Pharmacokinetic and anti-toxoplasma activity profiles of the printlets and compressed tablets were superimposable with no statistical difference (p > 0.05). CONCLUSION: Clinical performance of the printlets would be similar to the compressed tablets.

In-use stability assessment of FDA approved metformin immediate release and extended release products for N-Nitrosodimethylamine and dissolution quality attributes.

Metformin is a widely used first-line oral antidiabetic agent. TheFood and Drug Administration (FDA) confirmed the presence of the ofN-nitrosodimethylamine (NDMA) impurity, a carcinogenic, above the acceptable daily intake (ADI, 96 ng/day) in certain metformin products. The objective of the present study was to assess in-use stability of commercial metformin products for NDMA and dissolution quality attributes. Four immediate-release (M1-M4) and six extended-rerelease (M5-M10) metformin products were evaluated in the stability testing. All products were repacked in pharmacy vials and stored at 30 °C/75% RH for 12 weeks. Five products (M2, M3, M5, M7 and M10) had NDMA level above ADI limit (96 ng/day) before in-use stability exposure. NDMA in M2 (1164 ± 52.9 ng/tablet) and M3 (3776 ± 351.9 ng/tablet) products were 12 and 39 folds of ADI, respectively. Similarly, ER products, M5 (191 ± 94.1 ng/tablet), M7 (1473 ± 47.3 ng/tablet) and M10 (423 ± 55.8 ng/tablet) exhibited NDMA of 1.9, 15.3 and 4.4 folds of ADI, respectively. The impurity level significantly (p < 0.05) increased after 12-week stability exposure to 2.72, 2.47, 2.23 and 2.78 folds of initial values in M2, M3, M7 and M10. In summary, these findings suggested that carcinogenic impurity generation was affected by in-use stability condition exposure and it is expected that several more products currently in the market may also be recalled soon.

Therapeutic Application of Microsponges-based Drug Delivery Systems.

Microsponges delivery systems (MDS) are highly porous, cross-linked polymeric systems that activate due to temperature, pH, or when rubbed. MDS offer a wide range of advantages, like controlled drug release, site-specific action, stability over a broad range of pH, less irritation, cost-effectiveness, and improved patient compliance. They can be transformed into various dosage forms like creams, gels, and lotions. MDS are suitable for the treatment of topical disorders like acne, psoriasis, dandruff, eczema, scleroderma, hair loss, skin cancer, and other dreadful diseases. The applications of MDS in drug delivery are not limited to topical drug delivery but are also explored for oral, parenteral, and pulmonary drug deliveries. Microsponges have been studied for colon targeting of drugs and genes. Additionally, MDS have several applications such as sunscreen, cosmetics, and over-the-counter (OTC) products. Furthermore, MDS do not actuate any irritation, genotoxicity, immunogenicity, or cytotoxicity. Therefore, this review extensively highlights microsponges, their advantages, key factors affecting their characteristics, their therapeutic applications in topical disorders and in cancer, their use as cosmetics, as well as recent advances in MDS and the associated challenges.

Development and Validation Of A Discriminatory Dissolution Method for Portioned Moist Snuff and Snus.

Portioned moist snuff and snus, two subcategories of smokeless tobacco products (STP) were dissolution tested as a quality control test. A USP Apparatus 4 was employed to develop and validate the method. The method was assessed based on time to reach nicotine dissolution plateau, percentage difference between two profiles at each time point, relative standard deviation (RSD), and f1 (similarity) and f2 (dissimilarity) values. Based on these criteria, 200 ml volume and 8 ml/min flow were found be discriminatory. The amount of nicotine dissolved from the nine products varied widely (2.0-3.4, 2.1-4.1, 3.3-4.6, 5.5-6.6, 6.9-9.1, 11.5-14.2, 12.5-14.6, 14.0-15.5, and 15.5-19.6 mg/pouch at 60 min). RSDs of the dissolution ranges were more than 20% at earlier time points and less than 20% at later timepoints. The developed method produced distinct profiles for all the tested products, which was further confirmed by f1>15 and f2<50 values. In conclusion, the developed method was discriminatory and can be employed as a quality control test and to differentiate among moist snuff and snus products.

Potential Application of USP Paddle and Basket Dissolution Methods in Discriminating for Portioned Moist Snuff and Snus Smokeless Tobacco Products.

The focus of the study was to develop discriminatory dissolution methods for portioned snus and moist snuff sub-categories of smokeless tobacco products (STPs) using USP basket and paddle apparatuses. Skoal Classic Wintergreen (SCW) and CORESTA CRP1.1 pouches were used as test products. The dissolution was performed at 10, 20, 30, 40, and 50 rpm basket or paddle speed in 500 ml artificial saliva pH 6.8. The products were also characterized for assay, pH, particle size, and loss on drying. The dissolution profiles were evaluated for amount/% of nicotine dissolved, time to reach plateau, and profiles comparison by f2 and f1 factors. The nicotine assay was 13.3 ± 0.2 and 7.6 ± 0.1 mg/pouch for SCW and CRP1.1, respectively. The nicotine dissolved in 30 min from SCW and CRP1.1 were 38.4-81.8 and 37.6-88.1, and 50.5-64.9 and 72.3-92.1% by paddle and basket methods, respectively. The f2 and f1 values were ≤ 39.2 and ≥ 42.1 and ≤ 43.2 and ≥ 34.1 for basket methods and paddle methods. RSD were less than 20% at all points of dissolution profiles, and dissolution plateau were achieved in 30 min at some of the tested conditions. In summary, dissolution methods based on basket and paddle can be used as a performance test for STPs.

Lesson Learnt from Recall of Valsartan and Other Angiotensin II Receptor Blocker Drugs Containing NDMA and NDEA Impurities.

The presence of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities in angiotensin II receptor blocker (ARB) drugs containing tetrazole ring has triggered worldwide product recalls. The purpose of this article is to identify the potential gap area in current pharmaceutical industry practice that might have led to the NMDA and NDEA impurities escaping the drug manufacturer's and FDA's attention. The impact of process change was not adequately assessed by the manufacturer of contaminated APIs (active pharmaceutical ingredients), and potential for generation of mutagenic or other toxic impurities was not considered. The safety and risk associated with a chemical synthetic process was also not evaluated. This is primarily due to current industry practice which focuses on controlling the impurities above reporting threshold. ICH Q3A and FDA guidance on genotoxic and carcinogenic impurities in drug substances and products need to be integrated so that the ICH Q3A decision tree (attachment 3) begins by checking whether the synthetic process has been evaluated for the potential to generate toxic impurities. The compliance with ICH Q3A limits should be carried out only after the process has been determined to be safe without the risk of generating mutagenic and carcinogenic impurities.

A headspace-gas chromatography method for isopropanol determination in warfarin sodium products as a measure of drug crystallinity.

Coumadin® a nd s everal generic products of warfarin s odium (WS) contain the crystalline form (clathrate) in which WS and isopropanol (IPA) are associated in a 2:1 molar ratio. IPA is critical in maintaining the WS crystalline structure. Physicochemical properties of the drug and drug product may change when the crystalline drug transforms to amorphous form. A headspace-gas chromatography (HS-GC) method was developed and validated for IPA determination in the WS drug product. n-propanol (NPA) was used as internal standard and the method was validated for specificity, system suitability, linearity, accuracy, precision, range, limits of detection and quantification, and robustness. The method was specific, with good resolution between IPA and NPA peaks. Chromatographic parameters (retention time, IPA/NPA area ratio, tailing factor, theoretical plates, USP symmetry, capacity factor, selectivity and resolution) were consistent over three days of validation. The analytical method was linear from 2-200 µg mL-1 (0.1- 10 % IPA present in the drug product). LOD and LOQ were 0.1 and 2 µg mL-1, respectively. Accuracy at low (2 µg mL-1) and high (200 µg mL-1) IPA concentrations of the calibration curve was 103.3-113.3 and 98.9-102.2 % of the nominal value, resp. The validated method was precise, as indicated by the RSD value of less than 2 % at three concentration levels of the calibration curve. The method reported here was utilized to determine accurately and precisely the IPA content in in-house formulations and commercial products. In summary, IPA determination by HS-GC provides an indirect measure of WS crystallinity in the drug product. Nevertheless, it should be confirmed by another analytical method since IPA from the drug substance is not distinguishable from IPA that may be present outside the drug crystals in a dosage form when prepared by wet granulation with IPA.

Solid Matrix Based Lipidic Nanoparticles in Oral Cancer Chemotherapy: Applications and Pharmacokinetics.

Chemotherapeutic delivery by oral route in cancer patients has the potential to create "hospitalization free chemotherapy" which is a vision of oncologists, formulation scientists and patients. Such a therapeutic approach will improve patients' compliance, ease the burden of the patients' caregivers and significantly reduce the cost of treatment. In current clinical practice, chemotherapy carried out by intravenous injection or infusion leads to undesired side-effects such as plasma concentrations crossing the maximum safe concentration, rapid body clearance and lower bioavailability. Despite the presence of challenges such as poor aqueous solubility and stability of drugs and the presence of biological barriers like multidrug efflux transporter in the GI tract, oral cancer chemotherapy has the potential to surmount those obstacles. Lipid nanoparticles (LNPs) such as solid lipid nanoparticle, nanostructured lipid carriers, nano lipid-drug conjugates, mixed micelles, liposomes and nanoemulsions have shown some promising results for use in oral anticancer drug delivery through nanotechnological approach. LNPs demonstrate enhanced oral bioavailability owing to their ability to inhibit first pass metabolism via lymphatic absorption by chylomicron-linked and/or M-cell uptake. LNPs reduce the inter- and intrasubject pharmacokinetics variability of administrated drugs. Moreover, certain classes of phospholipids and surfactants used in the formulations of LNPs can suppress the P-glycoprotein efflux system. Here, we shall be discussing the biopharmaceutical challenges in oral cancer chemotherapy and how the LNPs may provide solutions to such challenges. The effect of GI tract environment on LNPs and pharmacokinetics shall also be discussed.

Tulsi oil as a potential penetration enhancer for celecoxib transdermal gel formulations.

The focus of the present study was to develop and evaluate the transdermal system of celecoxib. Transdermal gels composed of carbopol 940 in propylene glycol (PG) containing penetration enhancers. The formulations were characterized by permeation, pharmacokinetics, pharmacodynamics and histopathology. Celecoxib permeation across excised rat skins were statistically (p < 0.05) enhanced by tulsi oil compared to turpentine oil containing formulations. In comparison to orally administered formulations, the pharmacokinetic parameters of gel and control formulations were significantly higher (p < 0.05). The maximum plasma concentration (Cmax) obtained with formulations containing 4% turpentine and 6% tulsi oil was, respectively, 1.52 and 2.41 times higher than the formulations without penetration enhancer. Similarly, area under the curve (AUC) of these formulations was 1.70 and 2.40 times higher than the formulations without penetration enhancers. Anti-inflammatory studies demonstrated a statistically significant (p < 0.05) pharmacodynamics profile for the transdermal gel formulations compared to orally administered and control celecoxib formulations. Histopathological studies revealed some disruption in the epidermis without any toxic effect on the dermis layer of skin by penetration enhancers. In summary, the transdermal gel formulations of celecoxib containing penetration enhancers sustained drug level in the blood and will reduce the dose frequency as required with its conventional oral formulation.

Ion-pair chromatography for simultaneous analysis of ethionamide and pyrazinamide from their porous microparticles.

Ethionamide (ETA) and pyrazinamide (PZA) are considered the drugs of choice for the treatment of multidrug-resistant tuberculosis. Current methods available in the literature for simultaneous determination of ETA and PZA have low sensitivity or involve column modifications with lipophilic cations. The aim of this study was to develop a simple and validated reversed-phase ion-pair HPLC method for simultaneous determination of ETA and PZA for the characterization of polymeric-based porous inhalable microparticles in in vitro and spiked human serum samples. Chromatographic separation was achieved on a Phenomenex C18 column (250 mm × 4.6 mm) using a Shimadzu LC 10 series HPLC. The mobile phase consisted of A: 0.01% trifluoroacetic acid in distilled water and B: ACN/MeOH at 1:1 v/v. Gradient elution was run at a flow rate of 1.5 mL/min and a fixed UV wavelength of 280 nm. The validation characteristics included accuracy, precision, linearity, analytical range, and specificity. Calibration curves at seven levels for ETA and PZA were linear in the analytical range of 0.1-3.0 µg/mL with correlation coefficient of r (2) > 0.999. Accuracy for both ETA and PZA ranged from 94 to 106% at all quality control (QC) standards. The method was precise with relative standard deviation less than 2% at all QC levels. Limits of quantitation for ETA and PZA were 50 and 70 ng/mL, respectively. There was no interference from either the polymeric matrix ions or the biological matrix in the analysis of ETA and PZA.

Nanometric gold in cancer nanotechnology: current status and future prospect.

OBJECTIVES: This review elaborate on modified gold nanoparticulate concept in oncology, provides an overview of the use of gold nanoparticles in cancer treatment and discusses their potential applications and clinical benefits. KEY FINDINGS: Modified gold nanoparticles (e.g. rod, multipod and star or a hollow structure such as shell, box and cage) have promising applications in the fields of drug delivery and photothermal therapy in oncology due to their unique optical and photothermal properties and their ability to modify the surface and conjugate drugs/molecules with gold nanomaterial. Modified gold nanoparticles exhibit strong light absorption in the near-infrared region in which light can penetrate deeply into soft tissue. Moreover, recent advances have opened the way to site-specific delivery by gold nanoparticle. SUMMARY: Recent research and development in cancer-targeted gold nanovectors shows promise for maximizing the efficacy of anti-cancer drugs while decreasing their harmful systemic effects in chemotherapy. Moreover, gold nanoparticles can also serve as cancer therapeutic.

Nanomedicines as cancer therapeutics: current status.

As of 21st century, cancer is arguably the most complex and challenging disease known to mankind and an inevitable public health concern of this millennium. Nanotechnology, suitably amalgamated with cancer research, has ushered an era of highly personalized and safer medicines which can improve cancer diagnosis and therapy. A wide variety of nanomedicines are currently under investigation, including polymeric/non-polymeric nanoparticles, dendrimers, quantum dots, carbon nanotubes, lipid- and micelle-based nanoparticles. The bases of these nanomedicines in reducing toxicity associated with cancer therapy are their ability to carry a large payload and multivalent-ligand targeting. This imparts specificity for targeting the tissues as well as bypass resistance mechanisms. The major hurdles on these future medicines are potential toxicity of nanoparticles, which imposes the need of extensive regulatory evaluation before nanomedicines could be utilized as cancer therapeutics. This review highlights nanopharmaceuticals that have been investigated in oncology for various applications (diagnosis, therapeutic delivery and theranostics). It also discusses the effects of nano-sized materials on tissues/organ functions, the possibility of overcoming multi-drug resistance by using nanomedicines and their current clinical status.

Evaluation of anticancer drug-loaded nanoparticle characteristics by nondestructive methodologies.

The purpose of this study was to utilize near-infrared (NIR) spectroscopy and near-infrared chemical imaging (NIR-CI) as non-invasive techniques to evaluate the drug loading in letrozole-loaded PLGA nanoparticle formulations prepared by the emulsification-solvent evaporation method. A Plackett-Burman design was applied to evaluate the main effects of amount of drug (X(1)), amount of polymer (X(2)), stirring rate (X(3)), emulsifier concentration (X(4)), organic to aqueous phase volume ratio (X(5)), type of organic solvent (X(6)), and homogenization time (X(7)) on drug entrapment efficiency. The influence of three different spectral pretreatment methods (multiplicative scatter correction, standard normal variate, and Savitzky-Golay second derivative transformation with third-order polynomial) and two different regression methods (PLS regression and principal component regression (PCR)) on model prediction ability were compared. PLS of spectra that were pretreated with Savitzky-Golay second derivative transformation provided better model prediction than PCR as it revealed better linear correlation (correlation coefficient of 0.991) for both calibration and prediction models. Relatively low values of root mean square errors of calibration (RMSEC = 0.748) and prediction (RMSEP = 0.786) and low standard errors of calibration (SEC = 0.758) and prediction (SEP = 0.589) suggested good predictability for estimation of the loading of letrozole in PLGA nanoparticles. NIR-CI analysis also revealed mutual homogenous distribution of both polymer and drug and was capable of clearly distinguishing the 12 formulations both quantitatively and qualitatively. In conclusion, NIR and NIR-CI could be potentially used to characterize anticancer drug-loaded nanoparticulate matrix.

Improvement in bioavailability of transdermally applied flurbiprofen using tulsi (Ocimum sanctum) and turpentine oil.

Penetration enhancing potential of tulsi and turpentine oil on transdermal delivery of flurbiprofen, a potent non-steroidal anti-inflammatory agent, was investigated. The transdermal permeation rate of flurbiprofen across the rat abdominal skin from binary solvent mixture composition of propylene glycol (PG):isopropyl alcohol (IPA) (30:70%, v/v) was 98.88 microg/cm(2)/h, significantly higher than other binary solvent mixtures. The corresponding steady state plasma concentration, 0.71 microg/ml, was much lower than required steady state plasma concentration of 3-5 microg/ml. Hence influence of tulsi and turpentine oil in the optimized binary solvent mixture along with the increased drug load on the flurbiprofen permeation was evaluated. The magnitude of the flux enhancement factor with turpentine oil and tulsi oil was 2.4 and 2.0 respectively at 5% (v/v) concentration beyond which there was no significant increase in the flux. Addition of 2% (w/v) hydroxypropyl methylcellulose (HPMC), as a thickening agent, resulted in desired consistency for the fabrication of patch with insignificant effect on permeation rate of flurbiprofen. The reservoir type of transdermal patch formulation, fabricated by encapsulating the flurbiprofen reservoir solution within a shallow compartment moulded from polyester backing film and microporous ethyl vinyl acetate membrane, did not modulate the skin permeation of flurbiprofen through rat skin in case of turpentine formulations whereas flux of formulations with tulsi oil was significantly altered. The influence of penetration enhancer and solvents on the anatomical structure of the rat skin was studied. Enhancement properties exhibited by turpentine oil and tulsi oil in optimized binary solvent mixture were superior as compared to solvent treated and normal control groups with negligible skin irritation. The fabricated transdermal patches were found to be stable. The bioavailability of flurbiprofen with reference to orally administered flurbiprofen in albino rats was found to increase by 2.97, 3.80 and 5.56 times with transdermal patch formulation without enhancer, tulsi and turpentine oil formulations, respectively. The results were confirmed by pharmacodynamic studies in rat edema inflammation model.

In-vivo evaluation in rats of colon-specific microspheres containing 5-fluorouracil.

The aims of this investigation were to determine the distribution in the gastrointestinal (GI) tract of Eudragit S-100 encapsulated colon-specific sodium alginate microspheres containing 5-fluorouracil (5-FU) in rats, and to perform pharmacokinetic and pharmacodynamic studies. Comparisons were with a control immediate-release (IR) formulation of 5-FU. 5-FU was distributed predominantly in the upper GI tract from the IR formulation but was distributed primarily to the lower part of the GI tract from the microsphere formulation. No drug was released in the stomach and intestinal regions from the colon-specific microspheres. Significantly, a high concentration of the active drug was achieved in colonic tissues from the colon-specific microspheres (P < 0.001), which was higher than the IC50 required to halt the growth of and/or kill colon cancer cells. Colon cancer was induced in rats by subcutaneous injection of 1,2-dimethylhydrazine (40 mg kg (-1)) for 10 weeks. The tumours induced were non-invasive adenocarcinomas and were in Duke's stage A. The 5-FU formulations were administered for 4 weeks after tumour induction. Non-significant reductions in tumour volume and multiplicity were observed in animals given the colon-specific microspheres. Enhanced levels of liver enzymes (SGOT, SGPT and alkaline phosphatase) were found in animals given the IR formulation of 5-FU, and values differed significantly (P < 0.001) from those in animals treated with the colon-specific microspheres. Elevated levels of serum albumin and creatinine, and leucocytopenia and thrombocytopenia were observed in the animals given the IR formulation. In summary, Eudragit S-100 coated alginate microspheres delivered 5-FU to colonic tissues, with reduced systemic side-effects. A long-term dosing study is required to ascertain the therapeutic benefits.

Characterization of 5-fluorouracil microspheres for colonic delivery.

The purpose of this investigation was to prepare and evaluate the colon-specific microspheres of 5-fluorouracil for the treatment of colon cancer. Core microspheres of alginate were prepared by the modified emulsification method in liquid paraffin and by cross-linking with calcium chloride. The core microspheres were coated with Eudragit S-100 by the solvent evaporation technique to prevent drug release in the stomach and small intestine. The microspheres were characterized by shape, size, surface morphology, size distribution, incorporation efficiency, and in vitro drug release studies. The outer surfaces of the core and coated microspheres, which were spherical in shape, were rough and smooth, respectively. The size of the core microspheres ranged from 22 to 55 microm, and the size of the coated microspheres ranged from 103 to 185 microm. The core microspheres sustained the drug release for 10 hours. The release studies of coated microspheres were performed in a pH progression medium mimicking the conditions of the gastrointestinal tract. Release was sustained for up to 20 hours in formulations with core microspheres to a Eudragit S-100 coat ratio of 1:7, and there were no changes in the size, shape, drug content, differential scanning calorimetry thermogram, and in vitro drug release after storage at 40 degrees C/75% relative humidity for 6 months.